Analysis of phosphorylated metabolites in crayfish extracts by two- dimensional ¹H-³¹P NMR heteronuclear total correlation spectroscopy (heteroTOCSY)

In vivo and extract analyses by one-dimensional ³¹P NMR have been a key tool in investigating energy-related metabolism. Although many phosphorylated metabolites have been observed, many of them have yet to be identified. This reflects the difficulty in identifying them using ³¹P NMR alone. Two-dimensional ¹H-³¹P correlation experiments have been shown to be useful for assigning phosphorylated metabolites. To obtain better sensitivity and structure information, ¹H-detected ³¹P-¹H heteronuclear total correlation spectroscopy (heteroTOCSY) was implemented and a complete chemical shift assignment for a number of phosphorylated standards was made. The time courses of 1D heteroTOCSY signal intensity versus spin-locking time were established for these standards to aid the optimization of the 2D experiment. This method was applied to crayfish extracts for the assignment of glucose 6-phosphate, α-glycerophosphate, ribose 5-phosphate, fructose 1,6-bisphosphate, phosphocholine, phosphoethanolamine, glucose 1-phosphate, glycerophosphoethanolamine, glycerophosphocholine, ATP, ADP, and AMP. An alkyl phosphate, a hexose 1-phosphate, and a UDP-hexose were also observed. These assignments allowed the identification of many changes in the ³¹P MR spectra of crayfish extracts elicited by treatment with the organophosphate pesticide chlorpyrifos. The assignment of an in vivo ³¹P spectrum of a live crayfish was also made based on the extract assignment. This approach should be a powerful tool for examining stress-associated changes in the metabolism of phosphorylated compounds.